DNA methylation is a key heritable epigenetic mark that dictates gene expression profiles within a cell. DNA methylation patterning is particularly important in that a hallmark of cancer cells is aberrant hypermethylation causing repression of gene promoters, especially of tumor suppressor genes. Post-translational histone modifications are also important epigenetic marks that have been implicated in regulating DNA methylation patterning. Several known histone marks, including methylation of histone H3 at lysines 4 and 27 and ubiquitylation of histone H2A (H3K4me, H3K27me, and H2AK119ub, respectively), are believed to differentially regulate DNA methyltransferase activity on DNA. The histone mark H3K4me is associated with gene activation, and H3K27me and H2AK119ub are associated with loci of transcriptional repression and DNA methylation, especially at hypermethylated genes in cancer. Recent studies have examined the genome-wide distribution of these histone modifications or have studied how their distribution relates to DNA methylation within specific loci, but these studies have not ascribed specific roles to these histone modifications in DNA methylation patterning, nor have they explained why only subsets of genes throughout the genome become hypermethylated in cancer. This project's main goal is to characterize the functional relationship between these marks and DNA methylation on a genome-wide basis. This project will focus on two aims: (1) To investigate how H3K4me affects genome-wide DNA methylation patterning and transcriptional regulation; and (2) to determine how the transcriptionally repressive marks H3K27me and H2AK119ub affect genome-wide DNA methylation patterning and transcriptional regulation. These aims will be accomplished by using siRNA in tissue culture cells against key subunits of the different complexes that establish each of these histone modifications. Through these siRNA treatments, we will assess the roles of each mark in establishing DNA methylation patterns throughout the genome and how these patterns relate to genome-wide gene expression. The effects of histone mark deficiency will be analyzed by an assay that isolates methylated-CpG-containing DNA coupled to massively parallel DNA sequencing to identify sequences with altered methylation patterns. These sequencing results will be correlated with patterns of gene expression determined by microarray analysis. Independent regions and individual genes of interest will be confirmed by quantitative-reverse transcription-PCR and bisulfite pyrosequencing.